44 results on '"Marie-Helene Disatnik"'
Search Results
2. A selective inhibitor of mitofusin 1-βIIPKC association improves heart failure outcome in rats
- Author
-
Julio C. B. Ferreira, Juliane C. Campos, Nir Qvit, Xin Qi, Luiz H. M. Bozi, Luiz R. G. Bechara, Vanessa M. Lima, Bruno B. Queliconi, Marie-Helene Disatnik, Paulo M. M. Dourado, Alicia J. Kowaltowski, and Daria Mochly-Rosen
- Subjects
Science - Abstract
Abstract We previously demonstrated that beta II protein kinase C (βIIPKC) activity is elevated in failing hearts and contributes to this pathology. Here we report that βIIPKC accumulates on the mitochondrial outer membrane and phosphorylates mitofusin 1 (Mfn1) at serine 86. Mfn1 phosphorylation results in partial loss of its GTPase activity and in a buildup of fragmented and dysfunctional mitochondria in heart failure. βIIPKC siRNA or a βIIPKC inhibitor mitigates mitochondrial fragmentation and cell death. We confirm that Mfn1-βIIPKC interaction alone is critical in inhibiting mitochondrial function and cardiac myocyte viability using SAMβA, a rationally-designed peptide that selectively antagonizes Mfn1-βIIPKC association. SAMβA treatment protects cultured neonatal and adult cardiac myocytes, but not Mfn1 knockout cells, from stress-induced death. Importantly, SAMβA treatment re-establishes mitochondrial morphology and function and improves cardiac contractility in rats with heart failure, suggesting that SAMβA may be a potential treatment for patients with heart failure.
- Published
- 2019
- Full Text
- View/download PDF
3. Potential biomarkers to follow the progression and treatment response of Huntington’s disease
- Author
-
Blair R. Leavitt, Mehrdad Shamloo, Amit Joshi, Daria Mochly-Rosen, Xin Qi, Marie Helene Disatnik, and Nay L. Saw
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Huntingtin ,Immunology ,Disease ,Biology ,Bioinformatics ,DNA, Mitochondrial ,Article ,Mice ,Protein Aggregates ,03 medical and health sciences ,0302 clinical medicine ,Huntington's disease ,Biopsy ,medicine ,Huntingtin Protein ,Animals ,Humans ,Immunology and Allergy ,Muscle, Skeletal ,Research Articles ,Genetic testing ,Inflammation ,Aldehydes ,Behavior, Animal ,medicine.diagnostic_test ,Neurotoxicity ,Case-control study ,Brain ,medicine.disease ,Body Fluids ,Mitochondria ,3. Good health ,Huntington Disease ,Phenotype ,030104 developmental biology ,Case-Control Studies ,Disease Progression ,Oxidation-Reduction ,Biomarkers ,030217 neurology & neurosurgery ,DNA Damage - Abstract
Disatnik et al. identify mitochondrial DNA levels, 8-OHdG, and inflammation factors as potential peripheral biomarkers to follow progression and treatment response of Huntington’s disease., Huntington’s disease (HD) is a rare genetic disease caused by expanded polyglutamine repeats in the huntingtin protein resulting in selective neuronal loss. Although genetic testing readily identifies those who will be affected, current pharmacological treatments do not prevent or slow down disease progression. A major challenge is the slow clinical progression and the inability to biopsy the affected tissue, the brain, making it difficult to design short and effective proof of concept clinical trials to assess treatment benefit. In this study, we focus on identifying peripheral biomarkers that correlate with the progression of the disease and treatment benefit. We recently developed an inhibitor of pathological mitochondrial fragmentation, P110, to inhibit neurotoxicity in HD. Changes in levels of mitochondrial DNA (mtDNA) and inflammation markers in plasma, a product of DNA oxidation in urine, mutant huntingtin aggregates, and 4-hydroxynonenal adducts in muscle and skin tissues were all noted in HD R6/2 mice relative to wild-type mice. Importantly, P110 treatment effectively reduced the levels of these biomarkers. Finally, abnormal levels of mtDNA were also found in plasma of HD patients relative to control subjects. Therefore, we identified several potential peripheral biomarkers as candidates to assess HD progression and the benefit of intervention for future clinical trials.
- Published
- 2016
4. A selective inhibitor of mitofusin 1-βIIPKC association improves heart failure outcome in rats
- Author
-
Luiz Roberto Grassmann Bechara, Luiz H. M. Bozi, Daria Mochly-Rosen, Paulo Magno Martins Dourado, Juliane C. Campos, Marie-Helene Disatnik, Julio C.B. Ferreira, Nir Qvit, Alicia J. Kowaltowski, Vanessa Morais Lima, Bruno B. Queliconi, and Xin Qi
- Subjects
Male ,0301 basic medicine ,Programmed cell death ,Science ,Myocardial Infarction ,General Physics and Astronomy ,02 engineering and technology ,Mitochondrion ,Article ,General Biochemistry, Genetics and Molecular Biology ,GTP Phosphohydrolases ,Mitochondrial Proteins ,Contractility ,Gene Knockout Techniques ,03 medical and health sciences ,Protein Kinase C beta ,medicine ,Animals ,MFN1 ,Myocyte ,Myocytes, Cardiac ,Phosphorylation ,RNA, Small Interfering ,Rats, Wistar ,lcsh:Science ,Protein kinase C ,Heart Failure ,Multidisciplinary ,business.industry ,Cardiac myocyte ,Membrane Proteins ,General Chemistry ,ANATOMIA ,021001 nanoscience & nanotechnology ,medicine.disease ,Myocardial Contraction ,Cell biology ,030104 developmental biology ,Heart failure ,Mitochondrial Membranes ,lcsh:Q ,Peptides ,0210 nano-technology ,business - Abstract
We previously demonstrated that beta II protein kinase C (βIIPKC) activity is elevated in failing hearts and contributes to this pathology. Here we report that βIIPKC accumulates on the mitochondrial outer membrane and phosphorylates mitofusin 1 (Mfn1) at serine 86. Mfn1 phosphorylation results in partial loss of its GTPase activity and in a buildup of fragmented and dysfunctional mitochondria in heart failure. βIIPKC siRNA or a βIIPKC inhibitor mitigates mitochondrial fragmentation and cell death. We confirm that Mfn1-βIIPKC interaction alone is critical in inhibiting mitochondrial function and cardiac myocyte viability using SAMβA, a rationally-designed peptide that selectively antagonizes Mfn1-βIIPKC association. SAMβA treatment protects cultured neonatal and adult cardiac myocytes, but not Mfn1 knockout cells, from stress-induced death. Importantly, SAMβA treatment re-establishes mitochondrial morphology and function and improves cardiac contractility in rats with heart failure, suggesting that SAMβA may be a potential treatment for patients with heart failure., Beta II protein kinase C (βIIPKC) activation contributes to heart failure. Here the authors show, in a rat model of myocardial infarction, that heart failure outcome can be improved by selectively inhibiting the interaction between βIIPKC and its downstream mitochondrial target Mitofusin-1, and that this strategy is superior to global βIIPKC inhibition.
- Published
- 2019
5. New therapeutics to modulate mitochondrial dynamics and mitophagy in cardiac diseases
- Author
-
Daria Mochly-Rosen, Marie-Helene Disatnik, Sunhee Hwang, and Julio C.B. Ferreira
- Subjects
Fission ,Cardiotonic Agents ,Heart Diseases ,Mitochondrial Degradation ,Review ,030204 cardiovascular system & hematology ,Biology ,Mitochondrion ,Mitochondrial Dynamics ,Mitochondria, Heart ,Cardiac disease ,03 medical and health sciences ,0302 clinical medicine ,Drug Discovery ,Mitophagy ,Autophagy ,Animals ,Humans ,Genetics(clinical) ,Fusion ,Genetics (clinical) ,030304 developmental biology ,Medicine(all) ,0303 health sciences ,Heart ,Human genetics ,Mitochondria ,3. Good health ,Cell biology ,mitochondrial fusion ,MITOCÔNDRIAS ,Molecular Medicine - Abstract
The processes that control the number and shape of the mitochondria (mitochondrial dynamics) and the removal of damaged mitochondria (mitophagy) have been the subject of intense research. Recent work indicates that these processes may contribute to the pathology associated with cardiac diseases. This review describes some of the key proteins that regulate these processes and their potential as therapeutic targets for cardiac diseases.
- Published
- 2015
6. Cardioprotection induced by a brief exposure to acetaldehyde: role of aldehyde dehydrogenase 2
- Author
-
Rudá Prestes e Albuquerque, Daria Mochly-Rosen, Che-Hong Chen, Wenjin Yang, Marie-Helene Disatnik, Cintia B. Ueta, Juliane C. Campos, Angélica B. Sanchez, Marisa Helena Gennari de Medeiros, Vanessa Morais Lima, and Julio C.B. Ferreira
- Subjects
0301 basic medicine ,Male ,Time Factors ,Genotype ,Physiology ,Myocardial Infarction ,Aldehyde dehydrogenase ,Mice, Transgenic ,Myocardial Reperfusion Injury ,Acetaldehyde ,Pharmacology ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Enzyme activator ,Physiology (medical) ,Animals ,Humans ,Point Mutation ,Gene Knock-In Techniques ,Ethanol metabolism ,ALDH2 ,Cardioprotection ,biology ,Dose-Response Relationship, Drug ,Aldehyde Dehydrogenase, Mitochondrial ,Original Articles ,Cardiotoxicity ,Rats ,Enzyme Activation ,Mice, Inbred C57BL ,Dose–response relationship ,DOENÇAS CARDIOVASCULARES ,Disease Models, Animal ,030104 developmental biology ,Phenotype ,chemistry ,biology.protein ,Cardiology and Cardiovascular Medicine ,Ex vivo - Abstract
Aims We previously demonstrated that acute ethanol administration protects the heart from ischaemia/reperfusion (I/R) injury thorough activation of aldehyde dehydrogenase 2 (ALDH2). Here, we characterized the role of acetaldehyde, an intermediate product from ethanol metabolism, and its metabolizing enzyme, ALDH2, in an ex vivo model of cardiac I/R injury. Methods and results We used a combination of homozygous knock-in mice (ALDH2*2), carrying the human inactivating point mutation ALDH2 (E487K), and a direct activator of ALDH2, Alda-1, to investigate the cardiac effect of acetaldehyde. The ALDH2*2 mice have impaired acetaldehyde clearance, recapitulating the human phenotype. Yet, we found a similar infarct size in wild type (WT) and ALDH2*2 mice. Similar to ethanol-induced preconditioning, pre-treatment with 50 μM acetaldehyde increased ALDH2 activity and reduced cardiac injury in hearts of WT mice without affecting cardiac acetaldehyde levels. However, acetaldehyde pre-treatment of hearts of ALDH2*2 mice resulted in a three-fold increase in cardiac acetaldehyde levels and exacerbated I/R injury. Therefore, exogenous acetaldehyde appears to have a bimodal effect in I/R, depending on the ALDH2 genotype. Further supporting an ALDH2 role in cardiac preconditioning, pharmacological ALDH2 inhibition abolished ethanol-induced cardioprotection in hearts of WT mice, whereas a selective activator, Alda-1, protected ALDH2*2 against ethanol-induced cardiotoxicity. Finally, either genetic or pharmacological inhibition of ALDH2 mitigated ischaemic preconditioning. Conclusion Taken together, our findings suggest that low levels of acetaldehyde are cardioprotective whereas high levels are damaging in an ex vivo model of I/R injury and that ALDH2 is a major, but not the only, regulator of cardiac acetaldehyde levels and protection from I/R.
- Published
- 2017
7. Aldehyde dehydrogenase 2 activation and coevolution of its εPKC-mediated phosphorylation sites
- Author
-
Che-Hong Chen, Daria Mochly-Rosen, Leslie Cruz, Marie-Helene Disatnik, and Aishwarya Nene
- Subjects
0301 basic medicine ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Aldehyde dehydrogenase ,Oxidative phosphorylation ,Protein Kinase C-epsilon ,Isozyme ,Serine ,Evolution, Molecular ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Pharmacology (medical) ,Threonine ,Phosphorylation ,Molecular Biology ,Protein kinase C ,εPKC ,ALDH2 ,Biochemistry, medical ,biology ,Research ,Aldehyde Dehydrogenase, Mitochondrial ,Biochemistry (medical) ,Cell Biology ,General Medicine ,030104 developmental biology ,Biochemistry ,030220 oncology & carcinogenesis ,Aldehyde dehydrogenase 2 ,biology.protein ,4HNE ,Coevolution - Abstract
Background Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for the metabolism of many toxic aldehydes such as acetaldehyde, derived from alcohol drinking, and 4HNE, an oxidative stress-derived lipid peroxidation aldehyde. Post-translational enhancement of ALDH2 activity can be achieved by serine/threonine phosphorylation by epsilon protein kinase C (εPKC). Elevated ALDH2 is beneficial in reducing injury following myocardial infarction, stroke and other oxidative stress and aldehyde toxicity-related diseases. We have previously identified three εPKC phosphorylation sites, threonine 185 (T185), serine 279 (S279) and threonine 412 (T412), on ALDH2. Here we further characterized the role and contribution of each phosphorylation site to the enhancement of enzymatic activity by εPKC. Methods Each individual phosphorylation site was mutated to a negatively charged amino acid, glutamate, to mimic a phosphorylation, or to a non-phosphorylatable amino acid, alanine. ALDH2 enzyme activities and protection against 4HNE inactivation were measured in the presence or absence of εPKC phosphorylation in vitro. Coevolution of ALDH2 and its εPKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family. Results We identified S279 as a critical εPKC phosphorylation site in the activation of ALDH2. The critical catalytic site, cysteine 302 (C302) of ALDH2 is susceptible to adduct formation by reactive aldehyde, 4HNE, which readily renders the enzyme inactive. We show that phosphomimetic mutations of T185E, S279E and T412E confer protection of ALDH2 against 4HNE-induced inactivation, indicating that phosphorylation on these three sites by εPKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with εPKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, εPKC specific phosphorylation sites, while T412 is found in other ALDH isozymes. We further identified three highly conserved serine/threonine residues (T384, T433 and S471) in all 18 ALDH isozymes that may play an important phosphorylation-mediated regulatory role in this important family of detoxifying enzymes. Conclusion εPKC phosphorylation and its coevolution with ALDH2 play an important role in the regulation and protection of ALDH2 enzyme activity. Electronic supplementary material The online version of this article (doi:10.1186/s12929-016-0312-x) contains supplementary material, which is available to authorized users.
- Published
- 2017
8. Selective Phosphorylation Inhibitor of Delta Protein Kinase C-Pyruvate Dehydrogenase Kinase Protein-Protein Interactions: Application for Myocardial Injury in Vivo
- Author
-
Marie-Helene Disatnik, Nir Qvit, Daria Mochly-Rosen, and Eiketsu Sho
- Subjects
0301 basic medicine ,Male ,Pyruvate dehydrogenase kinase ,Myocardial Reperfusion Injury ,Mitogen-activated protein kinase kinase ,Protein Serine-Threonine Kinases ,Biochemistry ,Catalysis ,Article ,MAP2K7 ,Substrate Specificity ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,Colloid and Surface Chemistry ,Animals ,ASK1 ,Phosphorylation ,Rats, Wistar ,Protein kinase A ,Protein Kinase Inhibitors ,MAPK14 ,Mice, Inbred BALB C ,biology ,Chemistry ,Kinase ,Cyclin-dependent kinase 2 ,Pyruvate Dehydrogenase Acetyl-Transferring Kinase ,General Chemistry ,Recombinant Proteins ,Molecular Docking Simulation ,Protein Kinase C-delta ,030104 developmental biology ,030220 oncology & carcinogenesis ,biology.protein ,Peptides ,Protein Binding - Abstract
Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ∼50 nM and reducing cardiac injury IC50 of ∼5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors.
- Published
- 2016
9. Pharmacological inhibition of βIIPKC is cardioprotective in late-stage hypertrophy
- Author
-
Tomoyoshi Koyanagi, Patricia Chakur Brum, Giovanni Fajardo, Suresh S. Palaniyandi, Marie Helene Disatnik, Eric N. Churchill, Daria Mochly-Rosen, Daniel Bernstein, Julio C.B. Ferreira, and Grant R. Budas
- Subjects
Male ,medicine.medical_specialty ,chemistry.chemical_element ,Cardiomegaly ,Calcium ,Biology ,Sodium-Calcium Exchanger ,Article ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,Muscle hypertrophy ,Contractility ,Fibrosis ,Internal medicine ,Protein Kinase C beta ,Troponin I ,medicine ,Animals ,Myocytes, Cardiac ,Amino Acid Sequence ,Protein Kinase Inhibitors ,Molecular Biology ,Protein Kinase C ,Heart Failure ,Rats, Inbred Dahl ,Sodium-calcium exchanger ,Myocardium ,CARDIOLOGIA ,medicine.disease ,Myocardial Contraction ,Rats ,Disease Models, Animal ,Endocrinology ,chemistry ,Heart failure ,Myocardial fibrosis ,Cardiomyopathies ,Peptides ,Cardiology and Cardiovascular Medicine - Abstract
We previously found that in the hearts of hypertensive Dahl salt-sensitive rats, βIIPKC levels increase during the transition from compensated cardiac hypertrophy to cardiac dysfunction. Here we showed that a six-week treatment of these hypertensive rats with a βIIPKC-specific inhibitor, βIIV5-3, prolonged their survival by at least 6weeks, suppressed myocardial fibrosis and inflammation, and delayed the transition from compensated hypertrophy to cardiac dysfunction. In addition, changes in the levels of the Ca(2+)-handling proteins, SERCA2 and the Na(+)/Ca(2+) exchanger, as well as troponin I phosphorylation, seen in the control-treated hypertensive rats were not observed in the βΙΙPKC-treated rats, suggesting that βΙΙPKC contributes to the regulation of calcium levels in the myocardium. In contrast, treatment with the selective inhibitor of βIPKC, an alternative spliced form of βIIPKC, had no beneficial effects in these rats. We also found that βIIV5-3, but not βIV5-3, improved calcium handling in isolated rat cardiomyocytes and enhanced contractility in isolated rat hearts. In conclusion, our data using an in vivo model of cardiac dysfunction (late-phase hypertrophy), suggest that βIIPKC contributes to the pathology associated with heart failure and thus an inhibitor of βIIPKC may be a potential treatment for this disease.
- Published
- 2011
10. Aberrant mitochondrial fission in neurons induced by protein kinase Cδ under oxidative stress conditions in vivo
- Author
-
Ning Shen, Raymond A. Sobel, Marie Helene Disatnik, Daria Mochly-Rosen, and Xin Qi
- Subjects
Dynamins ,Male ,Programmed cell death ,Mitochondrion ,Biology ,Mitochondrial apoptosis-induced channel ,03 medical and health sciences ,0302 clinical medicine ,Tumor Cells, Cultured ,Animals ,Humans ,Amino Acid Sequence ,Phosphorylation ,Protein kinase A ,Molecular Biology ,030304 developmental biology ,Neurons ,0303 health sciences ,Rats, Inbred Dahl ,Cell Death ,Brain ,Cell Biology ,Articles ,Molecular biology ,Signaling ,Transport protein ,Mitochondria ,Protein Structure, Tertiary ,Rats ,Oxidative Stress ,Protein Kinase C-delta ,Protein Transport ,Multiprotein Complexes ,Hypertension ,DNAJA3 ,Mitochondrial fission ,Oligopeptides ,Sequence Alignment ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Impaired mitochondrial fusion/fission plays a causal role in neuronal death. This study delineated a PKCδ-related signaling cascade in which excessive mitochondrial fission is induced during oxidative stress. Moreover, a selective peptide inhibitor of PKCδ inhibits impaired mitochondrial fission under these pathological conditions., Neuronal cell death in a number of neurological disorders is associated with aberrant mitochondrial dynamics and mitochondrial degeneration. However, the triggers for this mitochondrial dysregulation are not known. Here we show excessive mitochondrial fission and mitochondrial structural disarray in brains of hypertensive rats with hypertension-induced brain injury (encephalopathy). We found that activation of protein kinase Cδ (PKCδ) induced aberrant mitochondrial fragmentation and impaired mitochondrial function in cultured SH-SY5Y neuronal cells and in this rat model of hypertension-induced encephalopathy. Immunoprecipitation studies indicate that PKCδ binds Drp1, a major mitochondrial fission protein, and phosphorylates Drp1 at Ser 579, thus increasing mitochondrial fragmentation. Further, we found that Drp1 Ser 579 phosphorylation by PKCδ is associated with Drp1 translocation to the mitochondria under oxidative stress. Importantly, inhibition of PKCδ, using a selective PKCδ peptide inhibitor (δV1-1), reduced mitochondrial fission and fragmentation and conferred neuronal protection in vivo and in culture. Our study suggests that PKCδ activation dysregulates the mitochondrial fission machinery and induces aberrant mitochondrial fission, thus contributing to neurological pathology.
- Published
- 2011
11. Mitochondrial import of PKCε is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury
- Author
-
Marie-Helene Disatnik, Grant R. Budas, Daria Mochly-Rosen, Lihan Sun, and Eric N. Churchill
- Subjects
Male ,Physiology ,Receptors, Cytoplasmic and Nuclear ,030204 cardiovascular system & hematology ,Mitochondrion ,Ischaemia reperfusion ,Mitochondria, Heart ,0302 clinical medicine ,Mitochondrial Precursor Protein Import Complex Proteins ,Protein kinase C epsilon ,Phosphorylation ,Inner mitochondrial membrane ,Cardioprotection ,0303 health sciences ,Aldehyde Dehydrogenase, Mitochondrial ,Mitochondria ,Transport protein ,Protein–protein interaction ,Protein Transport ,Mitochondrial Membranes ,Original Articles: Focus on Mitochondria in Cardiac Disease ,Cardiology and Cardiovascular Medicine ,Oligopeptides ,Protein Binding ,Translocase of the outer membrane ,Molecular Sequence Data ,Enzyme Activators ,Myocardial Reperfusion Injury ,Receptors, Cell Surface ,Protein Kinase C-epsilon ,Biology ,Mitochondrial Proteins ,Necrosis ,03 medical and health sciences ,Physiology (medical) ,Animals ,Humans ,Amino Acid Sequence ,HSP90 Heat-Shock Proteins ,Heat shock protein 90 ,Rats, Wistar ,Protein kinase C ,030304 developmental biology ,Myocardium ,Membrane Transport Proteins ,Recovery of Function ,Aldehyde Dehydrogenase ,Myocardial Contraction ,Molecular biology ,Rats ,Enzyme Activation ,Disease Models, Animal ,Cytosol ,Cytoprotection ,Drug Design ,Sequence Alignment - Abstract
Protein kinase C epsilon (PKCepsilon) is critical for cardiac protection from ischaemia and reperfusion (IR) injury. PKCepsilon substrates that mediate cytoprotection reside in the mitochondria. However, the mechanism enabling mitochondrial translocation and import of PKCepsilon to enable phosphorylation of these substrates is not known. Heat shock protein 90 (HSP90) is a cytoprotective protein chaperone that participates in mitochondrial import of a number of proteins. Here, we investigated the role of HSP90 in mitochondrial import of PKCepsilon.Using an ex vivo perfused rat heart model of IR, we found that PKCepsilon translocates from the cytosol to the mitochondrial fraction following IR. Immunogold electron microscopy and mitochondrial fractionation demonstrated that following IR, mitochondrial PKCepsilon is localized within the mitochondria, on the inner mitochondrial membrane. Pharmacological inhibition of HSP90 prevented IR-induced interaction between PKCepsilon and the translocase of the outer membrane (Tom20), reduced mitochondrial import of PKCepsilon, and increased necrotic cell death by approximately 70%. Using a rational approach, we designed a 7-amino acid peptide activator of PKCepsilon, derived from an HSP90 homologous sequence located in the C2 domain of PKCepsilon (termed psiepsilonHSP90). Treatment with this peptide (conjugated to the cell permeating TAT protein-derived peptide, TAT(47-57)) increased PKCepsilon-HSP90 protein-protein interaction, enhanced mitochondrial translocation of PKCepsilon, increased phosphorylation and activity of an intra-mitochondrial PKCepsilon substrate, aldehyde dehydrogenase 2, and reduced cardiac injury in ex vivo and in vivo models of myocardial infarction.Our results suggest that HSP90-mediated mitochondrial import of PKCepsilon plays an important role in the protection of the myocardium from IR injury.
- Published
- 2010
12. Ethanol for cardiac ischemia: the role of protein kinase c
- Author
-
Grant R. Budas, Daria Mochly-Rosen, Marie-Helene Disatnik, and Eric N. Churchill
- Subjects
Cirrhosis ,Myocardial Ischemia ,Ischemia ,Pharmacology ,Article ,chemistry.chemical_compound ,medicine ,Animals ,Humans ,Pharmacology (medical) ,Protein Kinase C ,Protein kinase C ,Ethanol ,business.industry ,Cardiac ischemia ,Acute ethanol ,Therapeutic effect ,Central Nervous System Depressants ,medicine.disease ,chemistry ,Anesthesia ,Ischemic Preconditioning, Myocardial ,Ischemic preconditioning ,Cardiology and Cardiovascular Medicine ,business - Abstract
The physiological effects of ethanol are dependent upon the amount and duration of consumption. Chronic excessive consumption can lead to diseases such as liver cirrhosis, and cardiac arrhythmias, while chronic moderate consumption can have therapeutic effects on the cardiovascular system. Recently, it has also been observed that acute administration of ethanol to animals prior to an ischemic event provides significant protection to the heart. This review focuses on the different modalities of chronic vs. acute ethanol consumption and discusses recent evidence for a protective effect of acute ethanol exposure and the possible use of ethanol as a therapeutic agent.
- Published
- 2008
13. Peptides Derived from the C2 Domain of Protein Kinase Cϵ (ϵPKC) Modulate ϵPKC Activity and Identify Potential Protein-Protein Interaction Surfaces
- Author
-
Marie-Helene Disatnik, Eric N. Churchill, Daria Mochly-Rosen, and Relly Brandman
- Subjects
chemistry.chemical_classification ,Activator (genetics) ,Peptide ,Cell Biology ,Biology ,Biochemistry ,Isozyme ,Transport protein ,chemistry ,Protein kinase A ,Molecular Biology ,Peptide sequence ,Protein kinase C ,C2 domain - Abstract
Peptides derived from protein kinase C (PKC) modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Souroujon, M. C., and Mochly-Rosen, D. (1998) Nat. Biotechnol. 16, 919-924). We previously demonstrated that the C2 domain of PKC plays a critical role in these interactions. By focusing on ϵPKC and using a rational approach, we then identified one C2-derived peptide that acts as an isozyme-selective activator and another that acts as a selective inhibitor of ϵPKC. These peptides were used to identify the role of ϵPKC in protection from cardiac and brain ischemic damage, in prevention of complications from diabetes, in reducing pain, and in protecting transplanted hearts. The efficacy of these two peptides led us to search for additional C2-derived peptides with PKC-modulating activities. Here we report on the activity of a series of 5-9-residue peptides that are derived from regions that span the length of the C2 domain of ϵPKC. These peptides were tested for their effect on PKC activity in cells in vivo and in an ex vivo model of acute ischemic heart disease. Most of the peptides acted as activators of PKC, and a few peptides acted as inhibitors. PKC-dependent myristoylated alanine-rich C kinase substrate phosphorylation in ϵPKC knock-out cells revealed that only a subset of the peptides were selective for ϵPKC over other PKC isozymes. These ϵPKC-selective peptides were also protective of the myocardium from ischemic injury, an ϵPKC-dependent function (Liu, G. S., Cohen, M. V., Mochly-Rosen, D., and Downey, J. M. (1999) J. Mol. Cell. Cardiol. 31, 1937-1948), and caused selective translocation of ϵPKC over other isozymes when injected systemically into mice. Examination of the structure of the C2 domain from ϵPKC revealed that peptides with similar activities clustered into discrete regions within the domain. We propose that these regions represent surfaces of protein-protein interactions within ϵPKC and/or between ϵPKC and other partner proteins; some of these interactions are unique to ϵPKC, and others are common to other PKC isozymes.
- Published
- 2007
14. Impaired GAPDH-induced mitophagy contributes to the pathology of Huntington's disease
- Author
-
Marie-Helene Disatnik, Daria Mochly-Rosen, and Sunhee Hwang
- Subjects
Pathology ,medicine.medical_specialty ,Programmed cell death ,Cell Survival ,Mitochondrial Degradation ,Nerve Tissue Proteins ,Mitochondrion ,Biology ,PC12 Cells ,Mice ,Huntington's disease ,stomatognathic system ,Mitophagy ,medicine ,Huntingtin Protein ,Animals ,Humans ,Nuclear protein ,Glyceraldehyde 3-phosphate dehydrogenase ,Research Articles ,glyceraldehyde-3-phosphate dehydrogenase ,Mice, Knockout ,Nuclear Proteins ,medicine.disease ,polyglutamine repeats ,Mitochondria ,Rats ,Huntington Disease ,Polyglutamic Acid ,biology.protein ,Molecular Medicine ,Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) ,Huntington’s disease - Abstract
Mitochondrial dysfunction is implicated in multiple neurodegenerative diseases. In order to maintain a healthy population of functional mitochondria in cells, defective mitochondria must be properly eliminated by lysosomal machinery in a process referred to as mitophagy. Here, we uncover a new molecular mechanism underlying mitophagy driven by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) under the pathological condition of Huntington’s disease (HD) caused by expansion of polyglutamine repeats. Expression of expanded polyglutamine tracts catalytically inactivates GAPDH (iGAPDH), which triggers its selective association with damaged mitochondria in several cell culture models of HD. Through this mechanism, iGAPDH serves as a signaling molecule to induce direct engulfment of damaged mitochondria into lysosomes (micro-mitophagy). However, abnormal interaction of mitochondrial GAPDH with long polyglutamine tracts stalled GAPDH-mediated mitophagy, leading to accumulation of damaged mitochondria, and increased cell death. We further demonstrated that overexpression of inactive GAPDH rescues this blunted process and enhances mitochondrial function and cell survival, indicating a role for GAPDH-driven mitophagy in the pathology of HD.
- Published
- 2015
15. Mitochondrial Reactive Oxygen Species at the Heart of the Matter: New Therapeutic Approaches for Cardiovascular Diseases
- Author
-
Che-Hong Chen, Daria Mochly-Rosen, Marie-Helene Disatnik, Opher S. Kornfeld, Nir Qvit, and Sunhee Hwang
- Subjects
Pathology ,medicine.medical_specialty ,Antioxidant ,Physiology ,medicine.medical_treatment ,Ros scavenger ,Mitochondrial Degradation ,Mitochondrion ,Biology ,Models, Biological ,Article ,Antioxidants ,Mitochondria, Heart ,medicine ,Animals ,Humans ,Protein kinase C ,chemistry.chemical_classification ,Reactive oxygen species ,Translation (biology) ,Oxidative Stress ,Biochemistry ,chemistry ,Cardiovascular Diseases ,Cardiology and Cardiovascular Medicine ,Reactive Oxygen Species ,Signal Transduction - Abstract
Reactive oxygen species (ROS) have been implicated in a variety of age-related diseases, including multiple cardiovascular disorders. However, translation of ROS scavengers (antioxidants) into the clinic has not been successful. These antioxidants grossly reduce total levels of cellular ROS including ROS that participate in physiological signaling. In this review, we challenge the traditional antioxidant therapeutic approach that targets ROS directly with novel approaches that improve mitochondrial functions to more effectively treat cardiovascular diseases.
- Published
- 2015
16. Elucidation of the role of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) interaction with mutant huntingtin (mtHtt) for mitochondrial elimination in Huntington's disease
- Author
-
Daria Mochly-Rosen, Marie-Helene Disatnik, and Sunhee Hwang
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Huntingtin ,biology ,Chemistry ,Mutant ,medicine.disease ,Biochemistry ,Molecular biology ,nervous system diseases ,nervous system ,Huntington's disease ,mental disorders ,Genetics ,biology.protein ,medicine ,Huntingtin Protein ,Molecular Biology ,Glyceraldehyde 3-phosphate dehydrogenase ,Biotechnology - Abstract
Huntington's disease (HD) is the major neurodegenerative disease caused by expanded polyglutamine (polyQ) repeats in the huntingtin protein (mutant huntingtin). The presence of the abnormal expansi...
- Published
- 2015
17. A caveolin-3 mutant that causes limb girdle muscular dystrophy type 1C disrupts Src localization and activity and induces apoptosis in skeletal myotubes
- Author
-
Marie-Helene Disatnik, Thomas A. Rando, Joshua C. Eby, and Gayle M. Smythe
- Subjects
Caveolin 3 ,Muscle Fibers, Skeletal ,Apoptosis ,Biology ,Caveolae ,Caveolins ,Muscular Dystrophies ,Myoblasts ,Mice ,Membrane Microdomains ,Caveolin ,medicine ,Animals ,Muscular dystrophy ,Muscle, Skeletal ,Lipid raft ,Cells, Cultured ,Myogenesis ,Cell Biology ,medicine.disease ,Cell biology ,src-Family Kinases ,Microscopy, Fluorescence ,Biochemistry ,Signal Transduction ,Proto-oncogene tyrosine-protein kinase Src ,Limb-girdle muscular dystrophy - Abstract
Caveolins are membrane proteins that are the major coat proteins of caveolae, specialized lipid rafts in the plasma membrane that serve as scaffolding sites for many signaling complexes. Among the many signaling molecules associated with caveolins are the Src tyrosine kinases, whose activation regulates numerous cellular functions including the balance between cell survival and cell death. Several mutations in the muscle-specific caveolin, caveolin-3, lead to a form of autosomal dominant muscular dystrophy referred to as limb girdle muscular dystrophy type 1C (LGMD-1C). One of these mutations (here termed the `TFT mutation') results in a deletion of a tripeptide (ΔTFT(63-65)) that affects the scaffolding and oligomerization domains of caveolin-3. This mutation causes a 90-95% loss of caveolin-3 protein levels and reduced formation of caveolae in skeletal muscle fibers. However, the effects of this mutation on the specific biochemical processes and cellular functions associated with caveolae have not been elucidated. We demonstrate that the TFT caveolin-3 mutation in post-mitotic skeletal myotubes causes severely reduced localization of caveolin-3 to the plasma membrane and to lipid rafts, and significantly inhibits caveolar function. The TFT mutation reduced the binding of Src to caveolin-3, diminished targeting of Src to lipid rafts, and caused abnormal perinuclear accumulation of Src. Along with these alterations of Src localization and targeting, there was elevated Src activation in myotubes expressing the TFT mutation and an increased incidence of apoptosis in those cells compared with control myotubes. The results of this study demonstrate that caveolin-3 mutations associated with LGMD-1C disrupt normal cellular signal transduction pathways associated with caveolae and cause apoptosis in muscle cells, all of which may reflect pathogenetic pathways that lead to muscle degeneration in these disorders.
- Published
- 2003
18. Sequential activation of individual PKC isozymes in integrin-mediated muscle cell spreading: a role for MARCKS in an integrin signaling pathway
- Author
-
Christine H. Lee, Daria Mochly-Rosen, Marie-Helene Disatnik, Thomas A. Rando, and Stéphane C. Boutet
- Subjects
Integrins ,Time Factors ,Integrin ,Biology ,Cell Line ,Focal adhesion ,Mice ,Cell Movement ,Cell Adhesion ,Animals ,Humans ,Phosphorylation ,MARCKS ,Muscle, Skeletal ,Myristoylated Alanine-Rich C Kinase Substrate ,Cell adhesion ,Protein Kinase C ,Protein kinase C ,Cell Size ,Integrin Signaling Pathway ,Calcium-Binding Proteins ,Intracellular Signaling Peptides and Proteins ,Membrane Proteins ,Cell Biology ,Protein-Tyrosine Kinases ,Phosphoproteins ,Actin cytoskeleton ,Cell biology ,Enzyme Activation ,Isoenzymes ,Focal Adhesion Kinase 1 ,Focal Adhesion Protein-Tyrosine Kinases ,biology.protein ,Signal transduction ,Glucosidases ,Signal Transduction - Abstract
To understand how muscle cell spreading and survival are mediated by integrins, we studied the signaling events initiated by the attachment of muscle cells to fibronectin (FN). We have previously demonstrated that muscle cell spreading on FN is mediated by alpha5beta1 integrin, is associated with rapid phosphorylation of focal adhesion kinase and is dependent on activation of protein kinase C (PKC). Here we investigated the role of individual PKC isozymes in these cellular processes. We show that alpha, delta and epsilonPKC are expressed in muscle cells and are activated upon integrin engagement with different kinetics - epsilonPKC was activated early, whereas alpha and deltaPKC were activated later. Using isozyme-specific inhibitors, we found that the activation of epsilonPKC was necessary for cell attachment to FN. However, using isozyme-specific activators, we found that activation of each of three isozymes was sufficient to promote the spreading of alpha5-integrin-deficient cells on FN. To investigate further the mechanism by which integrin signaling and PKC activation mediate cell spreading, we studied the effects of these processes on MARCKS, a substrate of PKC and a protein known to regulate actin dynamics. We found that MARCKS was localized to focal adhesion sites soon after cell adhesion and that MARCKS translocated from the membrane to the cytosol during the process of cell spreading. This translocation correlated with different phases of PKC activation and with reorganization of the actin cytoskeleton. Using MARCKS-antisense cDNA, we show that alpha5-expressing cells in which MARCKS expression is inhibited fail to spread on FN, providing evidence for the crucial role of MARCKS in muscle cell spreading. Together, the data suggest a model in which early activation of epsilonPKC is necessary for cell attachment; the later activation of alpha or deltaPKC may be necessary for the progression from attachment to spreading. The mechanism of PKC-mediated cell spreading may be via the phosphorylation of signaling proteins, such as MARCKS, that are involved in the reorganization of the actin cytoskeleton.
- Published
- 2002
19. Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling
- Author
-
Patricia Chakur Brum, Alicia J. Kowaltowski, Kátia Maria Sampaio Gomes, Marie-Helene Disatnik, Bruno B. Queliconi, Che-Hong Chen, Luiz Roberto Grassmann Bechara, Daria Mochly-Rosen, Vanessa Morais Lima, Juliane C. Campos, Paulo Magno, and Julio C.B. Ferreira
- Subjects
Cardiac function curve ,Male ,medicine.medical_specialty ,Physiology ,Ischemia ,Mitochondrion ,Biology ,Mitochondrial Proteins ,Physiology (medical) ,Internal medicine ,medicine ,INSUFICIÊNCIA CARDÍACA ,Animals ,Ventricular Function ,Myocytes, Cardiac ,Myocardial infarction ,Rats, Wistar ,Ventricular remodeling ,ALDH2 ,Heart Failure ,Ventricular Remodeling ,Aldehyde Dehydrogenase, Mitochondrial ,Aldehyde Dehydrogenase ,medicine.disease ,Myocardial Contraction ,Mitochondria ,Endocrinology ,Heart failure ,Cardiology ,Cardiology and Cardiovascular Medicine ,Reperfusion injury - Abstract
Aims We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects the heart against acute ischaemia/reperfusion injury. Here, we determined the benefits of chronic activation of ALDH2 on the progression of heart failure (HF) using a post-myocardial infarction model. Methods and results We showed that a 6-week treatment of myocardial infarction-induced HF rats with a selective ALDH2 activator (Alda-1), starting 4 weeks after myocardial infarction at a time when ventricular remodelling and cardiac dysfunction were present, improved cardiomyocyte shortening, cardiac function, left ventricular compliance and diastolic function under basal conditions, and after isoproterenol stimulation. Importantly, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodelling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, accumulation of 4-hydroxynonenal (4-HNE)-protein adducts and protein carbonyls seen in HF was not observed in Alda-1-treated rats, suggesting that increasing the activity of ALDH2 contributes to the reduction of aldehydic load in failing hearts. ALDH2 activation was associated with improved mitochondrial function, including elevated mitochondrial respiratory control ratios and reduced H2O2 release. Importantly, selective ALDH2 activation decreased mitochondrial Ca2+-induced permeability transition and cytochrome c release in failing hearts. Further supporting a mitochondrial mechanism for ALDH2, Alda-1 treatment preserved mitochondrial function upon in vitro aldehydic load. Conclusions Selective activation of mitochondrial ALDH2 is sufficient to improve the HF outcome by reducing the toxic effects of aldehydic overload on mitochondrial bioenergetics and reactive oxygen species generation, suggesting that ALDH2 activators, such as Alda-1, have a potential therapeutic value for treating HF patients.
- Published
- 2014
20. Engineering a delta protein kinase C inhibitor that selectively inhibits interaction and phosphorylation of one substrate of the multi‐substrate kinase (582.3)
- Author
-
Nir Qvit, Daria Mochly-Rosen, and Marie-Helene Disatnik
- Subjects
MAP kinase kinase kinase ,biology ,Chemistry ,Cyclin-dependent kinase 2 ,Mitogen-activated protein kinase kinase ,Biochemistry ,MAP2K7 ,Cell biology ,Genetics ,biology.protein ,Phosphorylation ,ASK1 ,Molecular Biology ,cGMP-dependent protein kinase ,Protein kinase C ,Biotechnology - Abstract
Rationale: Many kinases phosphorylate multiple substrates. To assess the role of an individual phosphorylation event without using laborious mutagenesis studies, selective pharmacological tools that inhibit phosphorylation of specific one substrate are needed. We focused on delta protein kinase C (δPKC) because δPKC activation after heart attack leads to cardiac damage. We reasoned that since pyruvate dehydrogenase kinase (PDK) phosphorylation by δPKC leads to inhibition of ATP production, it might be critical in the injurious effect δPKC after heart attack. To determine if of the many substrates of δPKC, PDK is the substrate that mediates this injurious effect in the heart, we designed a selective inhibitor of δPKC binding and phosphorylation of PDK. Methods: We designed a short peptide inhibitor that mimics a unique interaction between PDK and δPKC to selectively inhibit δPKC phosphorylation of PDK. This protein-protein interaction inhibitory peptide (PPIIP) was used in vitro, in cells and in intact hea...
- Published
- 2014
21. Protein folding creates structure-based, noncontiguous consensus phosphorylation motifs recognized by kinases
- Author
-
Deborah Schechtman, Felipe Augusto Nunes Ferraz, Helio Miranda Costa-Junior, Darlene Aparecida Pena, Tiago J. P. Sobreira, Denise Aparecida Berti, Munira Muhammad Abdel Baqui, José Xavier-Neto, Paulo S. L. Oliveira, Mariana Lemos Duarte, and Marie-Helene Disatnik
- Subjects
Threonine ,Protein Folding ,PKC Phosphorylation Site ,Amino Acid Motifs ,Green Fluorescent Proteins ,Biology ,Biochemistry ,PROTEÍNAS QUINASES ,Catalysis ,Substrate-level phosphorylation ,Tubulin ,Animals ,Humans ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Protein kinase C ,Protein Kinase C ,Kinase ,Lysine ,Phosphotransferases ,Cell Biology ,Cyclic AMP-Dependent Protein Kinases ,Molecular Docking Simulation ,A-site ,HEK293 Cells ,Mutation ,Mutagenesis, Site-Directed ,Protein folding ,Cattle ,HeLa Cells - Abstract
Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr 253 in α-tubulin as a site that is phosphorylated by protein kinase C βI (PKCβI). Thr 253 is not part of a linear PKC consensus motif. Instead, Thr 253 occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCβI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this “structurally formed” consensus motif and its importance for the protein kinase–substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.
- Published
- 2014
22. The challenge in translating basic research discoveries to treatment of Huntington disease
- Author
-
Marie-Helene Disatnik, Xin Qi, and Daria Mochly-Rosen
- Subjects
business.industry ,animal model ,Neurodegeneration ,Disease progression ,General Engineering ,neurodegeneration ,protein-protein interactions ,Disease ,Drp1 ,Huntington disease ,medicine.disease ,Addendum ,mitochondria ,P110 peptide inhibitor ,Animal model ,Basic research ,Huntingtin Protein ,Medicine ,Treatment strategy ,business ,Neuroscience ,polyglutamine - Abstract
Huntington disease is a rare neurodegenerative disease resulting from insertion and/or expansion of a polyglutamine repeats close to the N-terminal of the huntingtin protein. Although unequivocal genetic tests have been available for about 20 years, current pharmacological treatments do not prevent or slow down disease progression. Recent basic research identified potential novel drug targets for the treatment of Huntington disease. However, there are clear challenges in translating these discoveries into treatment strategies for these patients. The following is a brief discussion of these challenges using our recent experience as an example.
- Published
- 2013
23. Dystrophin mutations predict cellular susceptibility to oxidative stress
- Author
-
Thomas A. Rando, Marie-Helene Disatnik, and Jeffrey S. Chamberlain
- Subjects
musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,medicine.medical_specialty ,Mutation ,biology ,Physiology ,Myogenesis ,musculoskeletal system ,medicine.disease ,Protein oxidation ,medicine.disease_cause ,Cell biology ,Cellular and Molecular Neuroscience ,Endocrinology ,Physiology (medical) ,Internal medicine ,Utrophin ,medicine ,biology.protein ,Myocyte ,Neurology (clinical) ,Muscular dystrophy ,Dystrophin ,Oxidative stress - Abstract
Mutations in the dystrophin gene that lead to the expression of truncated forms of the dystrophin protein cause muscular dystrophies of varying severities both in humans and in mice. We have shown previously that dystrophin-deficient muscle is more susceptible to oxidative injury than is normal muscle. In this report, we have used muscle cells derived from mdx mice, which express no dystrophin, and mdx-transgenic strains that express full-length dystrophin or truncated forms of dystrophin to explore further the relationship between dystrophin expression and susceptibility of muscle to oxidative injury. We show that, when differentiated into myotubes, the relative susceptibility of the cell populations to oxidative stress correlates with the severity of the dystrophy in the strain from which the cells were isolated. The most susceptible populations exhibited the greatest oxidative damage as assessed by protein oxidation. Thus, the relative efficacy of truncated dystrophin proteins to protect muscle from necrotic degeneration in vivo is predicted by their ability to protect muscle cells from free radical mediated injury. These findings support the hypothesis that the dystrophin protein complex may have important regulatory or signaling properties in terms of cell survival and antioxidant defense mechanisms.
- Published
- 2000
24. Activation of Aldehyde Dehydrogenase-2 Reduces Ischemic Damage to the Heart
- Author
-
Marie Helene Disatnik, Che-Hong Chen, Daria Mochly-Rosen, Grant R. Budas, Thomas D. Hurley, and Eric N. Churchill
- Subjects
chemistry.chemical_classification ,Multidisciplinary ,biology ,business.industry ,Activator (genetics) ,Aldehyde dehydrogenase ,Cardiotonic Agents ,Pharmacology ,medicine.disease ,Enzyme activator ,Enzyme ,Biochemistry ,chemistry ,biology.protein ,Medicine ,Ischemic preconditioning ,Myocardial infarction ,business ,ALDH2 - Abstract
There is substantial interest in the development of drugs that limit the extent of ischemia-induced cardiac damage caused by myocardial infarction or by certain surgical procedures. Here, using an unbiased proteomic search, we identified mitochondrial aldehyde dehydrogenase 2 (ALDH2) as an enzyme whose activation correlates with reduced ischemic heart damage in rodent models. A high-throughput screen yielded a small-molecule activator of ALDH2 (Alda-1) that, when administered to rats before an ischemic event, reduced infarct size by 60%, most likely through its inhibitory effect on the formation of cytotoxic aldehydes. In vitro, Alda-1 was a particularly effective activator of ALDH2*2, an inactive mutant form of the enzyme that is found in 40% of East Asian populations. Thus, pharmacologic enhancement of ALDH2 activity may be useful for patients with wild-type or mutant ALDH2 who are subjected to cardiac ischemia, such as during coronary bypass surgery.
- Published
- 2008
25. Acute Inhibition of Excessive Mitochondrial Fission After Myocardial Infarction Prevents Long‐term Cardiac Dysfunction
- Author
-
Juliane C. Campos, Paulo Magno Martins Dourado, Julio C.B. Ferreira, Daria Mochly-Rosen, Kátia Maria Sampaio Gomes, Marie Helene Disatnik, and Xin Qi
- Subjects
FIS1 ,medicine.medical_specialty ,Time Factors ,cardiac myocytes ,Myocardial Infarction ,Ischemia ,Infarction ,Drp1 ,heart ,030204 cardiovascular system & hematology ,Mitochondrion ,Pharmacology ,Mitochondrial Dynamics ,Mitochondria, Heart ,GTP Phosphohydrolases ,Contractility ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Animals ,Medicine ,Myocytes, Cardiac ,Myocardial infarction ,Cells, Cultured ,Original Research ,030304 developmental biology ,Heart Failure ,0303 health sciences ,business.industry ,ANATOMIA ,medicine.disease ,Peptide Fragments ,Rats ,3. Good health ,mitochondria ,protein‐protein interaction inhibitor ,Cardiology ,Mitochondrial fission ,Cardiology and Cardiovascular Medicine ,business ,Ex vivo - Abstract
Background Ischemia and reperfusion ( IR ) injury remains a major cause of morbidity and mortality and multiple molecular and cellular pathways have been implicated in this injury. We determined whether acute inhibition of excessive mitochondrial fission at the onset of reperfusion improves mitochondrial dysfunction and cardiac contractility postmyocardial infarction in rats. Methods and Results We used a selective inhibitor of the fission machinery, P110, which we have recently designed. P110 treatment inhibited the interaction of fission proteins Fis1/Drp1, decreased mitochondrial fission, and improved bioenergetics in three different rat models of IR , including primary cardiomyocytes, ex vivo heart model, and an in vivo myocardial infarction model. Drp1 transiently bound to the mitochondria following IR injury and P110 treatment blocked this Drp1 mitochondrial association. Compared with control treatment, P110 (1 μmol/L) decreased infarct size by 28±2% and increased adenosine triphosphate levels by 70+1% after IR relative to control IR in the ex vivo model. Intraperitoneal injection of P110 (0.5 mg/kg) at the onset of reperfusion in an in vivo model resulted in improved mitochondrial oxygen consumption by 68% when measured 3 weeks after ischemic injury, improved cardiac fractional shortening by 35%, reduced mitochondrial H 2 O 2 uncoupling state by 70%, and improved overall mitochondrial functions. Conclusions Together, we show that excessive mitochondrial fission at reperfusion contributes to long‐term cardiac dysfunction in rats and that acute inhibition of excessive mitochondrial fission at the onset of reperfusion is sufficient to result in long‐term benefits as evidenced by inhibiting cardiac dysfunction 3 weeks after acute myocardial infarction.
- Published
- 2013
26. Differential Activation of Protein Kinase C Isozymes by Phorbol Ester and Collagen in Human Skin Microvascular Endothelial Cells
- Author
-
Lisa Y. Zhou, Marvin A. Karasek, Marie-Helene Disatnik, Daria Mochly-Rosen, and G. Scott Herron
- Subjects
Angiogenesis ,Dermatology ,Biology ,Isozyme ,Biochemistry ,angiogenesis ,chemistry.chemical_compound ,Phorbol Esters ,Humans ,Tissue Distribution ,human dermal microvascular endothelial cells ,Molecular Biology ,Cells, Cultured ,Protein Kinase C ,Protein kinase C ,Skin ,Kinase ,Microcirculation ,Cell Biology ,Cell biology ,Enzyme Activation ,Isoenzymes ,Endothelial stem cell ,chemistry ,Phorbol ,Collagen ,Endothelium, Vascular ,Wound healing ,Type I collagen - Abstract
Human dermal microvascular endothelial cells participate in activities including inflammation, wound healing, and angiogenesis (neovascularization). Two stages of angiogenesis can be mimicked in vitro by two models of cultured foreskin human dermal microvascular endothelial cells: the differentiation of epithelioid endothelial cells to spindle-shaped mesenchymal-like cells induced by phorbol ester treatment; and the formation of vascular channels induced by exposing the luminal surface of endothelial cell monolayers to type I collagen gels. The mechanisms underlying these two processes, however, are largely unknown. Protein kinase C isozymes, which are activated by phorbol esters, are important mediators in the angiogenic process. In the current work, we identified the protein kinase C isozymes present in human dermal microvascular endothelial cells and determined which of the isozymes are activated in response to phorbol ester or to collagen treatments. Using western blot analysis, we found that microvascular endothelial cells contain at least six protein kinase C isozymes (alpha, beta, delta, epsilon, zeta, eta). Immunocytochemical studies demonstrated that the isozymes are located in distinct cellular compartments and that following treatment with phorbol 12-myristate 13-acetate or with a collagen gel overlay, most isozymes (protein kinase C alpha, beta1, betaII, delta, epsilon, eta) translocated to different parts of the cell. Moreover, for two of these isozymes (betaII and eta), the localization differs after phorbol 12-myristate 13-acetate treatment as compared with collagen treatment. These results demonstrate that agents that mimic two stages in the angiogenic process in vitro initiate diverse changes in the subcellular localization of specific protein kinase C isozymes and suggest a role for different isozymes in this process.
- Published
- 1996
- Full Text
- View/download PDF
27. Stimulus-dependent Subcellular Localization of Activated Protein Kinase C; a Study with Acidic Fibroblast Growth Factor and Transforming Growth Factor- 1 in Cardiac Myocytes
- Author
-
Marie-Helene Disatnik, Daria Mochly-Rosen, and Shekira Nashone Jones
- Subjects
medicine.medical_treatment ,Blotting, Western ,Stimulation ,Biology ,Isozyme ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Cytosol ,Transforming Growth Factor beta ,medicine ,Animals ,Myocyte ,Molecular Biology ,Cells, Cultured ,Protein Kinase C ,Protein kinase C ,Cell Nucleus ,Myocardium ,Growth factor ,Subcellular localization ,Molecular biology ,Rats ,Enzyme Activation ,Isoenzymes ,Microscopy, Fluorescence ,chemistry ,Phorbol ,Fibroblast Growth Factor 1 ,Cardiology and Cardiovascular Medicine ,Transforming growth factor - Abstract
Protein kinase C (PKC) isozymes regulate a number of cardiac functions including contractility, gene expression, and hypertrophy. There are at least six PKC isozymes in neonatal rat ventricular myocytes. We have shown previously that stimulation of cardiac myocytes in culture with norepinephrine (NE) or phorbol 12-myristate 13-acetate (PMA) results in translocation of each isozyme to distinct subcellular sites. In the present work, we demonstrated that PKC isozymes vary in their sensitivity to stimulation by acidic fibroblast growth factor (aFGF) and transforming growth factor- β 1 (TGF- β 1). Moreover, immunocytochemical studies indicated differences in the subcellular localization of activated isozymes following stimulation with each growth factor. These data suggest that the site of translocation and the resulting function of individual PKC isozymes are distinct for different PKC activators. Identification of the PKC isozymes that respond to aFGF and TGF- β 1 and their subcellular localization may provide a molecular basis for the divergent cardiac functions mediated by these two growth factors.
- Published
- 1995
28. Interaction of protein kinase C with RACKI, a receptor for activated C-kinase: a role in β protein kinase C mediated signal transduction
- Author
-
Che-Hong Chen, B. L. Smith, Marie-Helene Disatnik, Daria Mochly-Rosen, and Dorit Ron
- Subjects
Receptors for Activated C Kinase ,MAP kinase kinase kinase ,Chemistry ,Receptor for activated C kinase 1 ,ASK1 ,Cyclin-dependent kinase 9 ,Mitogen-activated protein kinase kinase ,Biochemistry ,Protein kinase C ,Cell biology ,MAP2K7 - Published
- 1995
29. Developmental expression of protein kinase C subspecies in rat brain-pituitary axis
- Author
-
Marie-Helene Disatnik, Daria Mochly-Rosen, Rami Eyal, David Ben-Menahem, Moshe Kalina, Zvi Naor, Yael Marantz, Socorro Garcia-Navarro, and Nachum Reiss
- Subjects
Male ,Aging ,Pituitary gland ,medicine.medical_specialty ,Blotting, Western ,Central nervous system ,Biology ,Biochemistry ,Rats, Sprague-Dawley ,Endocrinology ,Western blot ,Internal medicine ,medicine ,Animals ,Rats, Wistar ,Molecular Biology ,Cells, Cultured ,Protein Kinase C ,Protein kinase C ,medicine.diagnostic_test ,Brain ,Immunohistochemistry ,Enzyme assay ,Rats ,Isoenzymes ,medicine.anatomical_structure ,Histone phosphorylation ,Pituitary Gland ,biology.protein ,Female ,Antibody - Abstract
We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKCα, PKCβ and PKCɛ when brains from 6-day-old and 3-month-old female rats were compared, whereas PKCτ and PKCδ increased markedly during this period. On the other hand, brain PKCζ decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKCα, PKCβ and PKCζ when 6-day-old and 3-month-old female rats were compared, while PKCτ was not detected. The major band of pituitary PKGδ (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change. On the other hand PKCɛ which appeared as one band (92 kDa) in the 6-day-old pituitaries, increased markedly in the 3-month-old rat and was followed by a second major band (68 kDa) which was not observed in the juvenile rat. The increase in the PKC-positive cells and the decrease in histone phosphorylation in the developing pituitary is probably due to the marked dominance of PKCɛ in the adult rat pituitary.
- Published
- 1994
30. Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C
- Author
-
Daria Mochly-Rosen, Marie-Helene Disatnik, S M Hernandez-Sotomayor, G Carpenter, and G Jones
- Subjects
Molecular Sequence Data ,Receptors, Cell Surface ,In Vitro Techniques ,Phospholipase ,Phospholipase C gamma ,Biology ,Receptors for Activated C Kinase ,Binding, Competitive ,Cell Line ,chemistry.chemical_compound ,Epidermal growth factor ,Humans ,Amino Acid Sequence ,Phosphorylation ,Protein Kinase C ,Protein kinase C ,Binding Sites ,Multidisciplinary ,Epidermal Growth Factor ,Tyrosine phosphorylation ,Cell biology ,Enzyme Activation ,Isoenzymes ,chemistry ,Biochemistry ,Type C Phospholipases ,Signal transduction ,Subcellular Fractions ,Research Article - Abstract
Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.
- Published
- 1994
31. Aldehyde dehydrogenase activator attenuates diabetic cardiomyopathy; a role in improving the quality of resident cardiac stem cells?
- Author
-
Daria Mochly-Rosen, Lihan Sun, Euan A. Ashley, Aleksandra Pavlovic, Suresh S. Palaniyandi, Marie-Helene Disatnik, Jyothi Jayaram Vishnumangalam, Vivek Bhalla, and Xiaoyu Xia
- Subjects
medicine.medical_specialty ,biology ,business.industry ,Activator (genetics) ,Aldehyde dehydrogenase ,medicine.disease ,Biochemistry ,Endocrinology ,Internal medicine ,Diabetic cardiomyopathy ,Genetics ,biology.protein ,Medicine ,Stem cell ,business ,Molecular Biology ,Biotechnology - Published
- 2010
32. Activation of aldehyde dehydrogenase 2 (ALDH2) confers cardioprotection in protein kinase C epsilon (PKCε) knockout mice
- Author
-
Marie-Helene Disatnik, Che-Hong Chen, Grant R. Budas, and Daria Mochly-Rosen
- Subjects
Protein Kinase C-epsilon ,Myocardial Ischemia ,Aldehyde dehydrogenase ,Biology ,Article ,Gene Expression Regulation, Enzymologic ,Mice ,medicine ,Animals ,Humans ,Molecular Biology ,Protein kinase C ,Protein Kinase C ,ALDH2 ,Cardioprotection ,Mice, Knockout ,Ethanol ,Aldehyde Dehydrogenase, Mitochondrial ,Heart ,Aldehyde Dehydrogenase ,medicine.disease ,Cytoprotection ,Cell biology ,Enzyme Activation ,Biochemistry ,Reperfusion Injury ,Knockout mouse ,Mutation ,biology.protein ,Cardiology and Cardiovascular Medicine ,Reperfusion injury ,Signal Transduction - Abstract
Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCvarepsilon). We recently identified aldehyde dehydrogenase 2 (ALDH2) as a PKCvarepsilon substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCvarepsilon knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCvarepsilon and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCvarepsilon, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCvarepsilon knockout hearts. Our data suggest that ALDH2 is downstream of PKCvarepsilon in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCvarepsilon to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.
- Published
- 2009
33. Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of varepsilonPKC and activation of aldehyde dehydrogenase 2
- Author
-
Daria Mochly-Rosen, Eric N. Churchill, and Marie-Helene Disatnik
- Subjects
Blotting, Western ,Ischemia ,Myocardial Ischemia ,Aldehyde dehydrogenase ,Myocardial Reperfusion Injury ,Protein Kinase C-epsilon ,Pharmacology ,medicine.disease_cause ,Mitochondria, Heart ,Article ,Medicine ,Immunoprecipitation ,Molecular Biology ,Heart metabolism ,ALDH2 ,biology ,Ethanol ,business.industry ,Aldehyde Dehydrogenase, Mitochondrial ,Biological Transport ,Aldehyde Dehydrogenase ,medicine.disease ,Oxidative Stress ,Biochemistry ,Ischemic Preconditioning, Myocardial ,biology.protein ,Ischemic preconditioning ,Creatine kinase ,Cardiology and Cardiovascular Medicine ,business ,Reperfusion injury ,Oxidative stress - Abstract
The cardioprotective effects of moderate alcohol consumption have been well documented in animal models and in humans. Protection afforded against ischemia and reperfusion injury (I/R) proceeds through an ischemic preconditioning-like mechanism involving the activation of epsilon protein kinase C (varepsilonPKC) and is dependent on the time and duration of ethanol treatment. However, the substrates of varepsilonPKC and the molecular mechanisms by which the enzyme protects the heart from oxidative damage induced by I/R are not fully described. Using an open-chest model of acute myocardial infarction in vivo, we find that intraperitoneal injection of ethanol (0.5 g/kg) 60 min prior to (but not 15 min prior to) a 30-minute transient ligation of the left anterior descending coronary artery reduced I/R-mediated injury by 57% (measured as a decrease of creatine phosphokinase release into the blood). Only under cardioprotective conditions, ethanol treatment resulted in the translocation of varepsilonPKC to cardiac mitochondria, where the enzyme bound aldehyde dehydrogenase-2 (ALDH2). ALDH2 is an intra-mitochondrial enzyme involved in the detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and 4-HNE mediates oxidative damage, at least in part, by covalently modifying and inactivating proteins (by forming 4-HNE adducts). In hearts subjected to I/R after ethanol treatment, the levels of 4-HNE protein adducts were lower and JNK1/2 and ERK1/2 activities were diminished relative to the hearts from rats subjected to I/R in the absence of ethanol. Together, this work provides an insight into the mitochondrial-dependent basis of ethanol-induced and varepsilonPKC-mediated protection from cardiac ischemia, in vivo.
- Published
- 2008
34. Regulation of Pax3 by proteasomal degradation of monoubiquitinated protein in skeletal muscle progenitors
- Author
-
Stéphane C. Boutet, Kevin Iori, Lauren S. Chan, Thomas A. Rando, and Marie-Helene Disatnik
- Subjects
Proteasome Endopeptidase Complex ,RAD23B ,PROTEINS ,Myoblasts, Skeletal ,Molecular Sequence Data ,DEVBIO ,Muscle Development ,DNA-binding protein ,General Biochemistry, Genetics and Molecular Biology ,Mice ,Ubiquitin ,medicine ,Monoubiquitination ,Animals ,Paired Box Transcription Factors ,Amino Acid Sequence ,Muscle, Skeletal ,PAX3 Transcription Factor ,Cells, Cultured ,biology ,Biochemistry, Genetics and Molecular Biology(all) ,Myogenesis ,Lysine ,Skeletal muscle ,PAX7 Transcription Factor ,musculoskeletal system ,Molecular biology ,Cell biology ,DNA-Binding Proteins ,medicine.anatomical_structure ,embryonic structures ,biology.protein ,Thermodynamics ,PAX7 ,Protein monoubiquitination ,Protein Processing, Post-Translational - Abstract
SummaryPax3 and Pax7 play distinct but overlapping roles in developmental and postnatal myogenesis. The mechanisms involved in the differential regulation of these highly homologous proteins are unknown. We present evidence that Pax3, but not Pax7, is regulated by ubiquitination and proteasomal degradation during adult muscle stem cell activation. Intriguingly, only monoubiquitinated forms of Pax3 could be detected. Mutation of two specific lysine residues in the C-terminal region of Pax3 reduced the extent of its monoubiquitination and susceptibility to proteasomal degradation, whereas introduction of a key lysine into the C-terminal region of Pax7 rendered that protein susceptible to monoubiquitination and proteasomal degradation. Monoubiquitinated Pax3 was shuttled to the intrinsic proteasomal protein S5a by interacting specifically with the ubiquitin-binding protein Rad23B. Functionally, sustained expression of Pax3 proteins inhibited myogenic differentiation, demonstrating that Pax3 degradation is an essential step for the progression of the myogenic program. These results reveal an important mechanism of Pax3 regulation in muscle progenitors and an unrecognized role of protein monoubiquitination in mediating proteasomal degradation.
- Published
- 2006
35. The susceptibility of muscle cells to oxidative stress is independent of nitric oxide synthase expression
- Author
-
David S. Bredt, Weiyun Zhuang, Josh C. Eby, Thomas A. Rando, Monica Cheong, Marie-Helene Disatnik, and Pramit K. Mohapatra
- Subjects
medicine.medical_specialty ,Physiology ,Cell ,Gene Expression ,Oxidative phosphorylation ,Nitric Oxide Synthase Type I ,Biology ,medicine.disease_cause ,Transfection ,Nitric oxide ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Mice ,Myofibrils ,Physiology (medical) ,Internal medicine ,medicine ,Myocyte ,Animals ,Muscular dystrophy ,Enzyme Inhibitors ,Muscle, Skeletal ,Cells, Cultured ,Mice, Knockout ,Dose-Response Relationship, Drug ,Cell Differentiation ,Muscular Dystrophy, Animal ,medicine.disease ,Oxidants ,Cell biology ,Nitric oxide synthase ,Mice, Inbred C57BL ,Disease Models, Animal ,Oxidative Stress ,medicine.anatomical_structure ,Endocrinology ,chemistry ,biology.protein ,Mice, Inbred mdx ,Neurology (clinical) ,Nitric Oxide Synthase ,Dystrophin ,Reactive Oxygen Species ,Oxidative stress - Abstract
The free radical, nitric oxide (NO·), has been implicated in the pathogenesis of muscular dystrophies because the enzyme, nitric oxide synthase (NOS), which produces NO·, binds to the dystrophin–glycoprotein complex (DGC). In various studies of tissue samples from human and animal muscular dystrophies due to DGC defects, correlations between reductions of NOS activity and disease severity have been reported. To test for any direct effect of NOS expression on muscle cell susceptibility, we examined muscle cells in vitro under conditions of experimentally altered NOS activity. There were no differences in susceptibility to oxidative stress between differentiated myotube cultures from wild-type and from neuronal NOS (nNOS)-deficient mice. Likewise, pharmacological inhibition of NOS did not alter cellular susceptibility to oxidative challenges. Overexpression of NOS neither enhanced nor diminished cellular susceptibility to oxidative stress. Finally, we assessed the effect of NOS overexpression on myotube cultures from dystrophin-deficient (mdx) mice. NOS protein was localized to both membrane and cytosolic compartments in the transduced cells. Still, no difference in susceptibility to oxidative stress was found between the NOS-overexpressing cells and control cells. These data suggest that muscle cell susceptibility to oxidative challenges is independent of the level of NOS expression. Therefore, any role NO· may play in the pathogenesis of muscular dystrophies is likely to be independent of its effect on the redox state of the cell. © 2001 John Wiley & Sons, Inc. Muscle Nerve 24: 502–511, 2001
- Published
- 2001
36. Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides
- Author
-
Thomas A. Rando, Marie-Helene Disatnik, and Lucy Z.-H. Zhou
- Subjects
musculoskeletal diseases ,mdx mouse ,congenital, hereditary, and neonatal diseases and abnormalities ,Molecular Sequence Data ,Muscle Fibers, Skeletal ,Gene Conversion ,Oligonucleotides ,Chimeraplasty ,Dystrophin ,Mice ,Utrophin ,medicine ,Animals ,Muscular dystrophy ,Muscle, Skeletal ,Multidisciplinary ,biology ,Base Sequence ,Oligonucleotide ,Chimera ,Reverse Transcriptase Polymerase Chain Reaction ,Point mutation ,Skeletal muscle ,Genetic Therapy ,Muscular Dystrophy, Animal ,Biological Sciences ,medicine.disease ,musculoskeletal system ,Molecular biology ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Gene Expression Regulation ,biology.protein ,Mice, Inbred mdx - Abstract
Chimeric RNA/DNA oligonucleotides (“chimeraplasts”) have been shown to induce single base alterations in genomic DNA both in vitro and in vivo . The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated “MDX1”) designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1–2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were “revertant fibers.” Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription–PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.
- Published
- 2000
37. Evidence of oxidative stress in mdx mouse muscle: studies of the pre-necrotic state
- Author
-
Yip Yu, Marie-Helene Disatnik, Jyotsna Dhawan, Michelle M Whirl, Thomas A. Rando, Alexa A Franco, and M. Flint Beal
- Subjects
musculoskeletal diseases ,medicine.medical_specialty ,mdx mouse ,Gene Expression ,medicine.disease_cause ,Nitric Oxide ,Nitric oxide ,chemistry.chemical_compound ,Mice ,Necrosis ,Reference Values ,Internal medicine ,medicine ,Myocyte ,Animals ,Muscular dystrophy ,biology ,Nitrotyrosine ,Muscles ,medicine.disease ,Nitric oxide synthase ,Mice, Inbred C57BL ,Oxidative Stress ,Endocrinology ,Neurology ,chemistry ,Biochemistry ,biology.protein ,Mice, Inbred mdx ,Tyrosine ,Neurology (clinical) ,Dystrophin ,Oxidoreductases ,Oxidative stress - Abstract
Considerable evidence indicates that free radical injury may underlie the pathologic changes in muscular dystrophies from mammalian and avian species. We have investigated the role of oxidative injury in muscle necrosis in mice with a muscular dystrophy due to a defect in the dystrophin gene (the mdx strain). In order to avoid secondary consequences of muscle necrosis, all experiments were done on muscle prior to the onset of the degenerative process (i.e. during the `pre-necrotic' phase) which lasted up to 20 days of age in the muscles examined. In pre-necrotic mdx muscle, there was an induction of expression of genes encoding antioxidant enzymes, indicative of a cellular response to oxidative stress. In addition, the levels of lipid peroxidation were greater in mdx muscle than in the control. Since the free radical nitric oxide (NO ⋅ ) has been shown to mediate oxidative injury in various disease states, and because dystrophin has been shown to form a complex with the enzyme nitric oxide synthase, we examined pre-necrotic mdx muscle for evidence of NO ⋅ -mediated injury by measuring cellular nitrotyrosine formation. By both immunohistochemical and electrochemical analyses, no evidence of increased nitrotyrosine levels in mdx muscle was detected. Therefore, although no relationship with NO ⋅ -mediated toxicity was found, we found evidence of increased oxidative stress preceding the onset of muscle cell death in dystrophin-deficient mice. These results lend support to the hypothesis that free radical-mediated injury may contribute to the pathogenesis of muscular dystrophies.
- Published
- 1999
38. Dystrophic muscle in mice chimeric for expression of alpha5 integrin
- Author
-
Richard O. Hynes, Joy T. Yang, Marie Helene Disatnik, Helen Rayburn, Thomas A. Rando, Daniela Taverna, and Roderick T. Bronson
- Subjects
muscular dystrophy ,Integrin ,Integrin alpha5 ,Muscular Dystrophies ,Cell Line ,Mice ,Antigens, CD ,medicine ,Myocyte ,Animals ,Muscular dystrophy ,Muscle, Skeletal ,Mice, Knockout ,biology ,Myogenesis ,Chimera ,chimeric mice ,Embryogenesis ,apoptosis ,Skeletal muscle ,Cell Biology ,medicine.disease ,Molecular biology ,Fibronectin ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Apoptosis ,biology.protein ,Female ,integrin α5β1 ,Regular Articles - Abstract
α5-deficient mice die early in embryogenesis (Yang et al., 1993). To study the functions of α5 integrin later in mouse embryogenesis and during adult life we generated α5 −/−;+/+ chimeric mice. These animals contain α5-negative and positive cells randomly distributed. Analysis of the chimerism by glucose- 6-phosphate isomerase (GPI) assay revealed that α5 −/− cells contributed to all the tissues analyzed. High contributions were observed in the skeletal muscle. The perinatal survival of the mutant chimeras was lower than for the controls, however the subsequent life span of the survivors was only slightly reduced compared with controls (Taverna et al., 1998). Histological analysis of α5 −/−;+/+ mice from late embryogenesis to adult life revealed an alteration in the skeletal muscle structure resembling a typical muscle dystrophy. Giant fibers, increased numbers of nuclei per fiber with altered position and size, vacuoli and signs of muscle degeneration–regeneration were observed in head, thorax and limb muscles. Electron microscopy showed an increase in the number of mitochondria in some muscle fibers of the mutant mice. Increased apoptosis and immunoreactivity for tenascin-C were observed in mutant muscle fibers. All the alterations were already visible at late stages of embryogenesis. The number of altered muscle fibers varied in different animals and muscles and was often increased in high percentage chimeric animals. Differentiation of α5 −/− ES cells or myoblasts showed that in vitro differentiation into myotubes was achieved normally. However proper adhesion and survival of myoblasts on fibronectin was impaired. Our data suggest that a novel form of muscle dystrophy in mice is α5-integrin-dependent.
- Published
- 1998
39. Muscle cells from mdx mice have an increased susceptibility to oxidative stress
- Author
-
Marie-Helene Disatnik, Alexa A Franco, Yip Yu, and Thomas A. Rando
- Subjects
musculoskeletal diseases ,Nitroprusside ,Paraquat ,congenital, hereditary, and neonatal diseases and abnormalities ,Programmed cell death ,Carbonyl Cyanide m-Chlorophenyl Hydrazone ,Necrosis ,Vitamin K ,Free Radicals ,Cell Survival ,Cellular differentiation ,Amidines ,Biology ,medicine.disease_cause ,Cell Fusion ,Dystrophin ,Mice ,Reference Values ,medicine ,Myocyte ,Animals ,Muscular dystrophy ,Muscle, Skeletal ,Genetics (clinical) ,Cells, Cultured ,Dose-Response Relationship, Drug ,Myogenesis ,Cell Differentiation ,Hydrogen Peroxide ,musculoskeletal system ,medicine.disease ,Oxidants ,Staurosporine ,Cell biology ,Mice, Inbred C57BL ,Oxidative Stress ,Neurology ,Pediatrics, Perinatology and Child Health ,Immunology ,biology.protein ,Mice, Inbred mdx ,Neurology (clinical) ,medicine.symptom ,Oxidative stress - Abstract
Several lines of evidence suggest that free radical mediated injury and oxidative stress may lead to muscle necrosis in the muscular dystrophies, including those related to defects in the dystrophin gene. We have examined muscle cell death using an in vitro assay in which the processes that lead to myofiber necrosis in vivo may be amenable to investigation in a simplified cell culture system. Using myotube cultures from normal and dystrophin-deficient (mdx) mice, we have examined the susceptibilities of the cells to different metabolic stresses. Dystrophin-deficient cells were more susceptible to free radical induced injury when compared to normal cells, but the two populations were equally susceptible to other forms of metabolic stress. The differential response appeared to be specifically related to dystrophin expression since undifferentiated myoblasts (which do not express dystrophin) from normal and mdx mice were equally sensitive to oxidative stress. Thus, the absence of dystrophin appears to render muscle specifically more susceptible to free radical induced injury. These results support the hypothesis that oxidative stress may lead to myofiber necrosis in these disorders. Elucidating the mechanisms leading to cell death may help to explain the variabilities in disease expression that are seen as a function of age, among different muscles, and across species in animals with muscular dystrophy due to dystrophin deficiency.
- Published
- 1998
40. The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor
- Author
-
Hongying Chen, John P. Murnane, Pius Brzoska, Nikki A. Levin, Michael F. Christman, Marie-Helene Disatnik, Daria Mochly-Rosen, and Yingfang Zhu
- Subjects
Molecular Sequence Data ,Vimentin ,Breast Neoplasms ,Chromatography, Affinity ,Cell Line ,Ataxia Telangiectasia ,Radiation, Ionizing ,medicine ,Tumor Cells, Cultured ,Animals ,Humans ,Amino Acid Sequence ,Protein kinase A ,Protein kinase C ,Cells, Cultured ,Protein Kinase C ,Cell Line, Transformed ,Leucine Zippers ,Multidisciplinary ,Binding Sites ,biology ,Sequence Homology, Amino Acid ,Kinase ,Genetic Complementation Test ,Zinc Fingers ,Transfection ,Cell cycle ,Fibroblasts ,medicine.disease ,Molecular biology ,DNA-Binding Proteins ,Ataxia-telangiectasia ,biology.protein ,Cattle ,Signal transduction ,Research Article ,Transcription Factors - Abstract
Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.
- Published
- 1995
41. Localization of protein kinase C isozymes in cardiac myocytes
- Author
-
Daria Mochly-Rosen, Marie-Helene Disatnik, and Giorgio Buraggi
- Subjects
Myocardium ,Fluorescent Antibody Technique ,Cell Biology ,Biology ,Subcellular localization ,Isozyme ,Cell biology ,Rats ,Enzyme Activation ,Isoenzymes ,Chemically defined medium ,chemistry.chemical_compound ,Norepinephrine ,Biochemistry ,chemistry ,Animals, Newborn ,Phorbol ,Myocyte ,Phosphorylation ,Animals ,Tetradecanoylphorbol Acetate ,Myofibril ,Protein kinase C ,Cells, Cultured ,Protein Kinase C - Abstract
Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell-soluble fraction to the cell-particulate fraction, presumably near their protein substrates. Therefore, identifying the subcellular localization of each activated PKC isozyme may help to elucidate its role in cardiac functions. In the present work, we have determined the subcellular localization of six PKC isozymes (alpha, beta I, beta II, delta, epsilon, and zeta) in nonstimulated cardiac myocytes and in myocytes stimulated by norepinephrine (2 microM) or phorbol 12-myristate 13-acetate (100 nM). Activated PKC isozymes were localized in various subcellular compartments such as inside the nucleus and on myofibrils. The presence of serum in the growth medium also caused a redistribution of PKC isozymes in the cells distinct from that obtained with cells cultured in defined medium. We suggest that isozyme-specific localization may determine phosphorylation of different protein substrates present at these respective translocation sites and the resulting PKC-mediated cellular responses.
- Published
- 1994
42. Effects of calcium and calcium-channel blocker methoxyverapamil on the beta-adrenoceptors in myocardial cells in vitro
- Author
-
Asher Shainberg and Marie-Helene Disatnik
- Subjects
endocrine system ,medicine.medical_specialty ,animal structures ,Time Factors ,medicine.drug_class ,chemistry.chemical_element ,Calcium channel blocker ,Calcium ,Biochemistry ,chemistry.chemical_compound ,Non-competitive inhibition ,Internal medicine ,Receptors, Adrenergic, beta ,medicine ,Cyclic AMP ,Animals ,Gallopamil ,Receptor ,Creatine Kinase ,Cells, Cultured ,Pharmacology ,Growth medium ,Myocardium ,Isoproterenol ,Heart ,In vitro ,Rats ,Dissociation constant ,Endocrinology ,Mechanism of action ,chemistry ,medicine.symptom - Abstract
The possible relationship between methoxyverapamil (D600) as a calcium-channel blocker and the beta-adrenoceptors was investigated on heart cells grown in culture, using [3H]CGP-12177 as a radioligand. Treatment with D600 (20 micrograms/mL) for 24 hr caused a decrease of 30% in the [3H]CGP-12177 binding sites. Scatchard analysis showed that the Bmax is similar in control and D600-treated cells, but the Kd in D600-treated cells increases. The effect of D600 on the isoproterenol-induced adenylate cyclase activation was examined and it was found that the D600 prevented the increase in cAMP obtained by isoproterenol treatment. These results indicate that the action of D600 on the beta-adrenoceptors is a competitive inhibition of the [3H]CGP-12177 binding sites. We investigated the effect of Ca2+ in the growth medium on the level of beta-adrenoceptors. Heart cells grown for 24 hr in Ca(2+)-free medium showed a decrease of 36% in the [3H]CGP-12177 binding sites without changing the dissociation constant. This decrease is probably a result of reduction in synthesis of the receptors. The level of receptors returned to control values following replenishment with normal growth medium. These results show that calcium is essential for the development of the beta-adrenoceptors in heart cells in vitro.
- Published
- 1992
43. Characterization of beta-adrenoceptors on rat skeletal muscle cells grown in vitro
- Author
-
Asher Shainberg, Sanford R. Sampson, and Marie-Helene Disatnik
- Subjects
medicine.medical_specialty ,Adrenergic beta-Antagonists ,Amiodarone ,Biology ,Ligands ,Biochemistry ,Binding, Competitive ,Propanolamines ,chemistry.chemical_compound ,Internal medicine ,Receptors, Adrenergic, beta ,medicine ,Radioligand ,Animals ,Binding site ,Receptor ,Cells, Cultured ,Pharmacology ,Skeletal muscle ,Muscle, Smooth ,Adrenergic beta-Agonists ,Ligand (biochemistry) ,Molecular biology ,In vitro ,Rats ,EGTA ,medicine.anatomical_structure ,Endocrinology ,chemistry ,Cell culture ,sense organs - Abstract
The binding properties of an hydrophilic β-adrenergic receptor radioligand, (−)[ 3 H](4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazolo-2-one); ([ 3 H]CGP-12177), were investigated in rat skeletal muscle cells in culture. The binding of [ 3 H]CGP-12177 at 25° was saturable, reversible and of high affinity ( K d = 1.3 ± 0.3 nM). The maximal number of [ 3 H]CGP-12177 binding sites was 30.6 ± 3.2 fmol/dish (34 ± 3.5 fmol/mg protein). β-Adrenergic agonists and antagonists inhibited [ 3 H]CGP-12177 binding. The competing ligand inhibition binding is a typical one for β 2 -adrenoceptors. The increase in β-adrenoceptors was independent of cell fusion. Amiodarone (10 −5 M) decreased the β-adrenoceptor number in skeletal muscle cells differentiated in vitro by 48%, while the affinity for [ 3 H]CGP-12177 was not affected.
- Published
- 1990
44. Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells
- Author
-
Zvi Nevo, Marie-Helene Disatnik, and Naphtali Savion
- Subjects
Male ,Glycoside Hydrolases ,Lymphoma ,Physiology ,Clinical Biochemistry ,Dermatan sulfate ,Extracellular matrix ,Cornea ,chemistry.chemical_compound ,Mice ,Extracellular ,medicine ,Animals ,Heparanase ,Chondroitin sulfate ,Endothelium ,Melanoma ,Cells, Cultured ,Glucuronidase ,biology ,Sulfates ,Macrophages ,Cell Biology ,Heparin ,Heparan sulfate ,Macrophage Activation ,Molecular biology ,carbohydrates (lipids) ,Kinetics ,chemistry ,Proteoglycan ,Biochemistry ,biology.protein ,Cattle ,medicine.drug - Abstract
Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16-BL6 melanoma; ESb T-lymphoma) attach, invade, and penetrate confluent vascular endothelial cell monlayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]O4 = -labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macrophages do not store the heparanase intracellularly but it is instead found pericellularly and requires a continuous cell-matrix contact at the optimal pH for maintaining cell growth. The degradation of [35S]O4 = -labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10 micrograms/ml), arteparon (10 micrograms/ml), and heparin at a concentration of 3 micrograms/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo-beta-glucuronidase D-saccharic acid 1,4-lactone failed to inhibit the degradation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. However, the following antiproteases--alpha 2-macroglobulin, antithrombin III, leupeptin, and phenylmethylsulfony fluoride (PMSF)--failed to inhibit this degradation process, and only alpha 1-antitrypsin inhibited the heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase, inhibited at concentrations of 1 and 3 micrograms/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.
- Published
- 1987
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.